Impaired urea synthesis and consequent hyperammonemia may occur secondary to inborn errors of the urea cycle, treatment with certain drugs or faulty hepatic fatty acid oxidation (FAO), fatty liver or "Metabolic Syndrome" (MS). The biochemical and metabolic mechanism(s) by which defective FAO and fatty liver impairs ureagenesis are not known. Nor is an effective treatment available. During the current funding period we found that agmatine (AGM), the product of arginine decarboxylase, elevates cellular [cAMP] and stimulates hepatic ureagenesis as well as FAO. Preliminary data demonstrate that AGM or 5-aminoimidazole-4-carboxamide-1-[unreadable]-D-ribofuranoside (AICAR), an activator of AMPK, enhances ureagenesis in a rat model of non-alcoholic fatty liver disease and in the obese Zucker rat, a model of MS. Hence, our overall aim in this renewal proposal is to elucidate the mechanism(s) by which AGM or AICAR regulates hepatic amino-N metabolism and urea synthesis in normal and disease states. A long-term objective is to develop a clinically applicable protocol to improve ureagenesis in cases of defective ureagenesis and ammonia detoxification. We propose to explore two Specific Aims/Hypotheses: (i) Mitochondrial dysfunction and decrease in N- acetylglutamate (NAG) synthesis and consequent failure of activation of carbamoyl phosphate synthetase-I (CPS-I) is an initial cause of impaired ureagenesis in defective FAO and fatty liver. AGM, which like AICAR, augments FAO, triggers a metabolic cascade that results in augmented urea synthesis and relieves the metabolic derangements associated with fatty liver and MS;and (ii) An alternative, but not mutually exclusive, hypothesis is that fatty liver decreases mitochondrial uptake and metabolism of glutamine. AGM promotes FAO, diminishes hepatic steatosis, and thus improves glutamine uptake and metabolism via phosphate dependent-glutaminase. Questions to be addressed include: (1) Is stimulation of ureagenesis by AGM mediated via phosphorylation and deactivation of acetyl-CoA carboxylase? (2) Is the AGM action dependent on activation of AMPK or the cAMP-PKA signaling pathway? and (3) Does AGM or AICAR increase the relative contribution of glutamine-N and/or blood ammonia for mitochondrial citrulline synthesis, perhaps secondary to an AGM-mediated elevation of cAMP? Experiments will be performed using a rat model of fatty liver and/or MS and various systems, including (a) a liver perfusion system;(b) isolated mitochondria;and (c) in vivo study. We will use 15N and/or 13C labeled precursors and state-of-the-art methodologies, including gas chromatography-mass spectrometry (GC-MS), MALDI-TOF-mass spectrometry, nuclear magnetic resonance (NMR) and techniques of molecular biology. The combination of these methodologies provides a superb tool to pinpoint the primary mechanism(s) of AGM or AICAR action. Data to be obtained will provide new and pivotal information to understand liver nitrogen metabolism in normal and disease states and thus may lead to development of a novel intervention to improve urea synthesis and detoxification of ammonia associated with fatty liver or in cases such as drug-induced inhibition of urea synthesis